This program is devoted to solving central questions in metallobiochemistry through the use of multinuclear, multifrequency (9, 35, 95 GHz) CW and pulsed electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) spectroscopies to characterize transition metal centers that are vital to human health and disease. With these techniques we are able to completely characterize the active-site environment of a biological metal ion, and do so for key catalytic intermediates trapped by rapid freeze-quench and cryoreduction methods. When combined with isotopic labeling enabled by methods of modern molecular biology, these techniques permit the use of ENDOR/ESEEM to structurally characterize every stage of an enzyme's catalytic cycle. The current grant period further has shown that novel kinetic protocols can augment such structural information about intermediates with fundamental information about their reactivity. Together, these techniques provide the means to determine in precise detail how metal centers function in biology. The specific aims of this program have expanded to include: formation of bioavailable nitrogen through enzymatic reduction of N2;anabolic and catabolic activation of O2 by heme and nonheme Fe enzymes;physiological protection against oxidative stress and contrasting toxicity, afforded by 'inorganic'Mn2+;enzymatic control of radical reactions in the 'Radical SAM'(S-adenosyl methionine) enzyme superfamily;development of new ENDOR methodologies and new applications in biology (Table 1). Table 1: Aims for the Coming Period. N2 Reduction by Nitrogenase: Intermediates and Mechanism Biomimetic Mo and Fe Complexes in vivo EPR/ENDOR of Mn2+: Protection from Oxidative Stress Mechanisms of Toxicity Dioxygen-activation by Fe enzymes: Heme Monooxygenases Nonheme Fe Enzymes Radical Reactions in Metalloenzymes:The "Radical SAM" Superfamily 'ENDOR Development: Hyperfine Signs Q-band Tubes at W Band. PUBLIC HEALTH RELEVANCE: Metal centers play a central role in human health and disease. Our program is devoted to the development and application of advanced paramagnetic resonance measurement techniques as a uniquely incisive means of revealing how biologically important metalcenters function. The systems to be studied play many essential roles, including:protection from oxidative stress and reactive-oxygen species;formation of the key signaling and immune-response molecule, nitric oxide;xenobiotic and drug metabolism;antibiotic formation and resistance;regulation of diabetes-related metabolic pathways;enzymatic control of metabolically vital free-radical reactions;enzymatic formation of the bio-available nitrogen on which two- thirds of the world's population depend.